Previous Recommended Dietary Allowances (RDAs) for vitamin C (ascorbate) and other water-soluble vitamins were based on preventing deficiency diseases with a margin of safety. We proposed that new RDAs for vitamins, with vitamin C as a model vitamin, could be determined using in situ kinetics, a concept developed by this laboratory. This proposal was adopted and expanded by the National Academy of Sciences as part of revised recommendations for vitamin C intake released in 2000. In situ kinetics has biochemical and clinical goals. The biochemical goals are to determine vitamin C molecular functions in relation to vitamin concentrations. For these studies we investigate vitamin C function in human tissues such as fibroblasts and neutrophils. To determine how intracellular concentration is regulated, two pathways of vitamin C accumulation were characterized. In one pathway, ascorbate is transported as such by carriers that are sodium-dependent, saturable, energy dependent, and inhibited by newly synthesized ascorbate analogs. The two human transporters hSVCT1 and hSVCT2 were cloned and characterized, and genomic characterization and studies of nucleotide polymorphisms are underway. In the second pathway, the extracellular oxidized form of vitamin C, dehydroascorbic acid, is accumulated as ascorbate in neutrophils by the process of ascorbate recycling. Dehydroascorbic acid is transported by glucose transporters GLUT I, III, and IV, and immediately reduced intracellularly to ascorbate by glutaredoxin (thioltransferase). Glutaredoxin from neutrophils was isolated, identified as the reducing activity, cloned, and characterized. Our studies show that vitamin C recycling occurs in neutrophils activated by bacteria, but specifically in neutrophils and not in bacteria. Studies are ongoing to characterize potential roles of vitamin C in neutrophils, including oxidant quenching, bacterial killing, and regulation of neutrophil apoptosis. Overall findings suggest that vitamin C function can be determined in relation to its concentration in living tissues. The clinical goals of in situ kinetics are to determine how vitamin concentrations are achieved in normal humans as a function of dose and whether achieved concentrations have functional consequences. An extensive clinical study was completed in healthy male inpatients hospitalized at the Clinical Center. For the first time, the following were described: the relationship between vitamin C doses over a wide range and its concentration in plasma and tissues; true bioavailability of vitamin C; vitamin urinary excretion in relation to dose; and potential adverse effects in relation to dose. Also for the first time, data for these parameters were obtained in healthy women, and the findings published in August 2001. Based on our data for men, RDAs for vitamin C were revised upward in 2000 by the National Academy of Sciences. For men the RDA in the United States and Canada was increased from 60 to 90 mg daily, and for women the RDA was increased from 60 to 75 mg daily. Based on our data, the following countries have also increased their RDAs for vitamin C: Germany, Austria, Denmark, France, Japan, and China. Because the known health benefits from vitamin C are from foods containing the vitamin, we recommend that vitamin C intake is from at least 5 servings of fruits and vegetables daily. Forthcoming molecular and clinical data from our laboratory may have further impact on vitamin C intake recommendations for healthy and ill people.